Reduction of tgf beta signaling in myeloid cells in the treatment of cancer

ABSTRACT

Methods of inhibiting metastasis in cancer patients are provided, wherein the methods comprise reducing TGFβ signaling, for example, by reducing TGF receptor II expression in myeloid cells. Vectors comprising a TGFβ receptor II RNAi nucleic acid sequence operably linked to a myeloid specific promoter also are provided. A method of diagnosing cancer in an individual by determining TGFβ receptor II expression in myeloid cells in the individual is provided. Additionally, a method of modulating TGFβ activity in myeloid cells in a cancer patient comprising administering a regulator of at least one of the GSK 3  and PI 3 K pathways to the patient is provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims the benefit of U.S. Provisional PatentApplication No. 61/525,025 filed Aug. 18, 2011, which is incorporated byreference.

BACKGROUND OF THE INVENTION

Conventional cancer therapeutic approaches, including radiation andchemotherapy, are nonselective and damage normal cells. Gene therapieshave exhibited limited success. This likely is because the vector orvirus inefficiently reaches the targeted tumor and/or the agents used ingene therapy interact with normal cells and to yield adverse effects(McCormick, Nat. Rev. Cancer, 1: 130-141 (2001), and Scanlon, AnticancerRes., 24: 501-504 (2004)).

TGFβ targeted therapies, such as neutralizing antibodies, smallmolecular inhibitors, and adenoviruses have been used in preclinical andclinical settings (Dumont et al., Cancer Cell, 3: 531-536 (2003)).However, TGFβ is well known to work as a tumor suppressor in early stagetumorigenesis and as a tumor promoter in later stages of tumorprogression (Yang et al., Cancer Res., 68: 9107-9111 (2008), and Yang etal, Trends Immunol., 31: 220-227 (2010). The underlying mechanisms forthis switch in function are not clear and pose a great challenge forTGFβ targeted therapies. This challenge in TGFβ targeted therapyrepresents a general problem of cancer biology, as many cancer relatedmolecules demonstrate a dual role of pro- and anti-cancer properties.

Thus, there remains a need for effective and specific treatment ofcancer for both animals and humans.

BRIEF SUMMARY OF THE INVENTION

The invention provides a method of inhibiting metastasis in a cancerpatient comprising reducing TGFβ receptor II expression in myeloid cellsin the cancer patient.

The invention also provides a method of inhibiting metastasis in acancer patient comprising (a) removing bone marrow comprising myeloidcells from the cancer patient, (b) reducing TGFβ signaling in themyeloid cells of the bone marrow ex vivo to yield TGFβsignaling-deficient bone marrow, and (c) administering the TGFβsignaling-deficient bone marrow to the cancer patient, so as to inhibitmetastasis in the cancer patient.

Additionally, the invention provides a method of inhibiting metastasisin a cancer patient comprising transplanting TGFβ receptor II(Tgfbr2)-deficient myeloid cells to the cancer patient.

The invention further provides a vector comprising a Tgfbr2 RNAi nucleicacid sequence operably linked to a myeloid specific promoter.

The invention provides a method of diagnosing cancer in an individualcomprising (a) obtaining a sample comprising myeloid cells from theindividual; and (b) determining TGFβ receptor II expression in themyeloid cells, wherein increased TGFβ receptor II expression in themyeloid cells relative to a control indicates a diagnosis of cancer inthe individual.

The invention also provides a method of modulating TGFβ activity inmyeloid cells in a cancer patient comprising administering a regulatorof at least one of the GSK3 and PI3K pathways to the patient.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIGS. 1A and 1B are graphs with percent expression (y-axis) of immunecells from spleen (sp), thymus (thy), bone marrow (bm), and lymph node(ln) (x-axis) for foxed control Tgfbr2^(flox/flox) (white bar) andTgfbr2^(MyeKo) (black bar) mice. Expression of CD4+ T cells, CD8+ Tcells, NKT cells, NK cells, B cells, MDSCs (Gr-1+CD11b+), andmacrophages are represented.

FIG. 2 is a graph with the number of 4T1 lung metastases (y-axis) fromfoxed control Tgfbr2^(flox/flox) (white bar) and Tgfbr2^(MyeKo) (blackbar) mice that received injection of 4Ti mammary tumor cells into the #2mammary fat pad (MFP) (x-axis).

FIG. 3 is a graph with the number of 4T1 lung metastases (y-axis) forfoxed control Tgfbr2^(flox/flox) (white bar) and Tgfbr2^(MyeKo) (blackbar) mice that received a tail vein injection of 5×10⁵ 4T1 tumor cells(x-axis)

FIG. 4 is a graph with the number of 3LL Lewis lung metastases on they-axis for floxed control Tgfbr2^(flox/flox) (white bar) andTgfbr2^(MyeKo) (black bar) mice that received tail vein injection of 3LLLewis lung cancer cells (x-axis).

FIG. 5 is a schematic showing the experimental design for adoptivetransfer of bone marrow to wild-type tumor-bearing mice. 4T1 cells(5×10⁵) were injected into mammary fat pad of wild-type mice on day 0(D0). The tumors were surgically removed on day 15 (D15), and the micewere left to recover until day 34 (D34) after tumor injection (allowingdevelopment of tumor invasion and metastasis). On D34, mice received abone marrow transplant from foxed control Tgfbr2^(flox/flox) orTgfbr2^(MyeKO) mice. Lung metastasis was examined on day 63 (D63).

FIGS. 6A and 6B are graphs demonstrating the differences in survival andthe number of lung metastases between floxed control Tgfbr2^(flox/flox)() (n=9) or Tgfbr2^(MyeKO) mice (▪) (n=8). FIG. 6A has percent survivalon the y-axis and days after tumor injection on the x-axis. FIG. 6B hasthe number of lung metastases on the y-axis for floxed controlTgfbr2^(flox/flox) (white bar) or Tgfbr2^(MyeKO) (black bar) mice.

FIG. 7 is a graph showing the fold changes in Gr-1+CD11b+ myeloid cell(MDSC) production (y-axis) in spleen, bone marrow, and peripheral bloodfor normal (white bar) and 4T1 tumor bearing (D35 tu) (black bar) mice.

FIG. 8 is a graph showing the percentage of CD33+CD34+CD15+ cells(y-axis) for healthy donors () and lung cancer patients (▪). Flowcytometry analysis of immature myeloid cells was performed withanti-CD33, CD34, and CD15 antibodies.

FIGS. 9A and 9B are graphs showing the density of PF4 expression(y-axis) in myeloid cells (9A) and in the premetastatic lungs (9B) offoxed control Tgfbr2^(flox/flox) (white bar) and Tgfbr2^(MyeKo) (blackbar) mice.

FIGS. 9C and 9D are graphs demonstrating the differences between lungmetastases and tumor weight (g) between wild-type (▪) and CXCR3 knockout() mice that received injection of 4Ti mammary tumor cells into the #2mammary fat pad. FIG. 9C has the number of lung metastasis on they-axis. FIG. 9D has tumor weight (g) on the y-axis.

FIGS. 10A and 10B are graphs showing relative expression of cytokines inGr-1+CD11b+ cells of floxed control Tgfbr2^(flox/flox) (white bar) orTgfbr2^(MyeKO) (black bar) mice. FIG. 10A is a graph with relativeexpression (y-axis) for each of arginase, INOS, VEGF, TNFα, MMP9, IL4,IL-10, IL12, and IFNγ (x-axis). FIG. 10B is a graph with relativeexpression (y-axis) for each of IL4, IL5, IL6, IL-10, IL13, IFNγ, andIL2 (x-axis) bar.

FIG. 11A is a graph showing the percentage of IFNγ positive CD8+ T cellsin the spleen of tumor-bearing Tgfbr2^(MyeKO) (black) mice compared toTgfbr2^(flox/flox) (white bar) mice.

FIG. 11B is a graph showing the number of IFNγ-producing cells detectedby ELISPOT in the spleens of Tgfbr2^(MyeKO) (black bar) andTgfbr2^(flox/flox) (white bar) mice.

FIGS. 11C and 11D are graphs demonstrating that IFNγ neutralizationincreased lung metastases (11C) and tumor size (11D) in bothTgfbr2^(flox/flox) mice. Mice were inoculated with 5×10⁴ 4T1 cells inthe #2 MFP. The mice were treated with IFNγ neutralizing antibody (1 mgon day 1, 3, and 6) or IgG control (0.5 mg on day 9, 12, 15, 18, 21, 24,and 27) through intraperiotoneal injection. For evaluation of lungmetastases, mice were euthanized on day 28 after tumor injection.

FIG. 11E is a graph demonstrating TGFβ1 level (pg/mL) in myeloid cells(Gr-1+CD11b+) of wild-type (Nor), tumor-bearing Tgfbr2^(MyeKO), andTgfbr2^(flox/flox) mice on the y-axis. Sorted Gr-1+CD11b+ cells werecultured overnight and supernatants were collected for TGFβ1 ELISA.

FIG. 12A depicts fluorescence analysis and magnetic cell sorting (MACS)of human immature myeloid cells (CD33+CD34+CD15+) before (left panel)and after (right panel) sorting.

FIG. 12B depicts the results of Western blot analysis of TβRIIexpression in CD33+CD34+CD15+ myeloid cells, wherein β-actin served as apositive control. Samples from two normal individuals and six laterstage lung cancer patients were examined.

DETAILED DESCRIPTION OF THE INVENTION

The inventors discovered that myeloid TGFβ signaling is an essentialpart of the tumor promoting role of TGFβ. Specifically, the inventorsdiscovered that the expression of TGFβ receptor II in myeloid cells oftumor hosts plays an essential role in metastasis. Increased TGFβsignaling in myeloid cells constitutes a critical part of thetumor-promoting role of TGFβ. When TGFβ receptor II is deleted inmyeloid cells, there is a significant decrease in tumor metastasis.

Accordingly, the invention provides a method of inhibiting metastasis ina cancer patient comprising reducing TGFβ signaling in the cancerpatient. TGFβ signaling can be reduced by any suitable method known inthe art, but is preferably reduced by reducing TGFβ receptor IIexpression in myeloid cells of the cancer patient.

The cancer patient can be any suitable patient, such as an animal (e.g.,mouse, rat, guinea pig, rabbit, hamster, cat, dog, horse, cow, pig,simian, or human) with cancer or who is at risk for cancer.

Non-limiting examples of specific types of cancers include cancer of thehead and neck, eye, skin, mouth, throat, esophagus, chest, bone, lung,colon, sigmoid, rectum, stomach, prostate, breast, ovaries, kidney,liver, pancreas, brain, intestine, heart or adrenals. More particularly,cancers include solid tumor, sarcoma, carcinomas, fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, Kaposi's sarcoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma, retinoblastoma, a blood-born tumor, acutelymphoblastic leukemia, acute lymphoblastic B-cell leukemia, acutelymphoblastic T-cell leukemia, acute myeloblastic leukemia, acutepromyelocytic leukemia, acute monoblastic leukemia, acuteerythroleukemic leukemia, acute megakaryoblastic leukemia, acutemyelomonocytic leukemia, acutenonlymphocyctic leukemia, acuteundifferentiated leukemia, chronic myelocytic leukemia, chroniclymphocytic leukemia, hairy cell leukemia, or multiple myeloma. See,e.g., Harrison's Principles of Internal Medicine, Eugene Braunwald etal., eds., pp. 491 762 (15th ed. 2001).

In one aspect of the invention, metastasis in a cancer patient isinhibited by (a) removing bone marrow comprising myeloid cells from thecancer patient, (b) reducing TGFβ signaling in the myeloid cells of thebone marrow ex vivo to yield TGFβ signaling-deficient bone marrow, and(c) administering the TGFβ signaling-deficient bone marrow to the cancerpatient, so as to inhibit metastasis in the cancer patient. The TGFβsignaling deficiency preferably is a result of decreased TGFβ receptorII expression.

TGFβ receptor II is a member of the Ser/Thr protein kinase family andthe TGFβ receptor subfamily. The encoded protein is a transmembraneprotein that has a protein kinase domain, forms a heterodimeric complexwith another receptor protein, and binds TGFβ. This receptor/ligandcomplex phosphorylates proteins, which then enter the nucleus andregulate the transcription of a subset of genes related to cellproliferation. Alternatively spliced transcript variants encodingdifferent isoforms have been characterized (GenBank Accession Nos.NM_(—)001024847 and NM_(—)003242).

TGFβ receptor II expression can be reduced by any suitable method, suchas RNA interference (RNAi), mutation of the nucleic acid sequenceencoding TGFβ receptor II (to produce a non-functional TGFβ receptorII), or knockout (deletion of the native gene or portion thereof) of thenucleic acid sequence encoding TGFβ receptor II. Alternatively, TGFβreceptor II activity can be reduced by inhibition of the polypeptide,for example, by administration of TGFβ receptor II-specific antibodies,peptidomimetics, or small molecules.

In another embodiment, TGFβ and/or TGFβ receptor II activity can bemediated (e.g., reduced) by administration of a modifier (e.g.,inhibitor or enhancer) of a member of a related signaling pathway (e.g.,the GSK3 and PI3K signaling pathways). For example, Example 7 describesthe use of a PI3K inhibitor (LY294002), which can be used to target ormodify myeloid cells in a cancer patient.

Accordingly, the invention also provides a method of modulating TGFβand/or TGF receptor II activity in myeloid cells in a cancer patientcomprising administering a regulator of at least one of the GSK3 andPI3K pathways to the patient. For example, the myeloid cells of thecancer patient can be removed from the cancer patient, contacted with aPI3K inhibitor or GSK3 enhancer, which results in a reduction in TGFβand/or TGFβ receptor II activity in the myeloid cells, and thenreintroduced into the cancer patient. TGFβ and/or TGFβ receptor IIactivity in the myeloid cells includes, but is not limited to, TGFβregulation of type 2 cytokines (e.g., IL-10 and IL-4) and PF4 in themyeloid cells.

In one aspect, the invention provides a vector comprising or consistingof a nucleotide sequence that encodes an RNAi agent, which is an RNAmolecule that is capable of RNA interference. Such RNA molecules arereferred to as siRNA (short interfering RNA that is a short-lengthdouble-stranded RNA, including, for example, a short hairpin RNA). Thenucleotide sequence that encodes the RNAi agent preferably hassufficient complementarity with a cellular nucleotide sequence of TGFβreceptor II to be capable of inhibiting the expression of TGFβ receptorII.

The siRNA can comprise an antisense code DNA coding for the antisenseRNA directed against a region of the TGFβ receptor II gene mRNA and/or asense code DNA coding for the sense RNA directed against the same regionof the TGFβ receptor II gene mRNA. The siRNA can be any suitable length,such as 15-50 (e.g., 20, 25, 30, 35, 40, or 45) nucleotides.

Alternatively, the RNAi agent can be an antisense RNA, which is an RNAstrand having a sequence complementary to the TGFβ receptor II genemRNA. Antisense RNA induces RNAi by binding to the TGFβ receptor II genemRNA. The antisense RNA can be any suitable length, such as 15-50 (e.g.,20, 25, 30, 35, 40, or 45) nucleotides.

The vector comprising or consisting of a nucleotide sequence thatencodes an RNAi agent can be any suitable vector, such as a viralvector, a plasmid, a yeast, a nanoparticle, or naked DNA. Suitable viralvectors, included poxviruses (e.g., orthopox viruses, such as vacciniaviruses, and avian poxviruses, such as fowlpox virus and canarypoxvirus), adenoviruses, adeno-associated viruses, and retroviruses.

The nucleotide sequence that encodes an RNAi agent can be operablylinked to a promoter (in the vector). The promoter preferably is amyeloid-specific promoter so that expression of the RNAi agent isspecific to myeloid cells. The CD11b promoter and the c-fes promoter areexamples of myeloid-specific promoters.

The RNAi agent, vector, or regulator of at least one of the GSK3 andPI3K pathways can be administered alone or in a composition (e.g.,pharmaceutical composition) that can comprise at least one carrier(e.g., a pharmaceutically acceptable carrier), as well as othertherapeutic agents. The RNAi agent, vector, or regulator or therespective composition can be administered by any suitable route,including parenteral, topical, oral, or local administration.

The composition (e.g., pharmaceutical composition) can comprise morethan one compound or composition of the invention. Alternatively, or inaddition, the composition (e.g., pharmaceutical composition) cancomprise one or more other pharmaceutically active agents or drugs.Examples of such other pharmaceutically active agents or drugs that maybe suitable for use in the composition (e.g., pharmaceuticalcomposition) include anticancer agents. Suitable anticancer agentsinclude, without limitation, alkylating agents; nitrogen mustards;folate antagonists; purine antagonists; pyrimidine antagonists; spindlepoisons; topoisomerase inhibitors; apoptosis inducing agents;angiogenesis inhibitors; podophyllotoxins; nitrosoureas; cisplatin;carboplatin; interferon; asparginase; tamoxifen; leuprolide; flutamide;megestrol; mitomycin; bleomycin; doxorubicin; irinotecan; and taxol,geldanamycin (e.g., 17-AAG), and various anti-cancer peptides andantibodies.

The carrier can be any of those conventionally used and is limited onlyby physio-chemical considerations, such as solubility and lack ofreactivity with the active compound(s), and by the route ofadministration. The pharmaceutically acceptable carriers describedherein, for example, vehicles, adjuvants, excipients, and diluents, arewell-known to those skilled in the art and are readily available to thepublic. It is preferred that the pharmaceutically acceptable carrier beone which is chemically inert to the active agent(s) and one which hasno detrimental side effects or toxicity (other than that desired by theactive compounds) under the conditions of use.

The choice of carrier will be determined in part by the particularcompound or composition of the invention and other active agents ordrugs used, as well as by the particular method used to administer thecompound and/or composition. Accordingly, there are a variety ofsuitable formulations of the composition (e.g., pharmaceuticalcomposition) of the inventive methods. The following formulations fororal, aerosol, parenteral, subcutaneous, intravenous, intramuscular,interperitoneal, rectal, and vaginal administration are exemplary andare in no way limiting. One skilled in the art will appreciate thatthese routes of administering the compound of the invention are known,and, although more than one route can be used to administer a particularcompound, a particular route can provide a more immediate and moreeffective response than another route.

Injectable formulations are among those formulations that are preferredin accordance with the invention. The requirements for effectivepharmaceutical carriers for injectable compositions are well-known tothose of ordinary skill in the art (See, e.g., Pharmaceutics andPharmacy Practice, J.B. Lippincott Company, Philadelphia, Pa., Bankerand Chalmers, eds., pages 238-250 (1982), and ASHP Handbook onInjectable Drugs, Toissel, 4th ed., pages 622-630 (1986)).

Topical formulations are well-known to those of skill in the art. Suchformulations are particularly suitable in the context of the inventionfor application to the skin.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the compound or compositiondissolved in diluents, such as water, saline, or orange juice; (b)capsules, sachets, tablets, lozenges, and troches, each containing apredetermined amount of the active ingredient, as solids or granules;(c) powders; (d) suspensions in an appropriate liquid; and (e) suitableemulsions. Liquid formulations may include diluents, such as water andalcohols, for example, ethanol, benzyl alcohol, and the polyethylenealcohols, either with or without the addition of a pharmaceuticallyacceptable surfactant. Capsule forms can be of the ordinary hard- orsoft-shelled gelatin type containing, for example, surfactants,lubricants, and inert fillers, such as lactose, sucrose, calciumphosphate, and corn starch. Tablet forms can include one or more oflactose, sucrose, mannitol, corn starch, potato starch, alginic acid,microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicondioxide, croscarmellose sodium, talc, magnesium stearate, calciumstearate, zinc stearate, stearic acid, and other excipients, colorants,diluents, buffering agents, disintegrating agents, moistening agents,preservatives, flavoring agents, and pharmacologically compatibleexcipients. Lozenge forms can comprise the active ingredient in aflavor, usually sucrose and acacia or tragacanth, as well as pastillescomprising the active ingredient in an inert base, such as gelatin andglycerin, or sucrose and acacia, emulsions, gels, and the likecontaining, in addition to the active ingredient, such excipients as areknown in the art.

The compounds and compositions of the invention, alone or in combinationwith other suitable components, can be made into aerosol formulations tobe administered via inhalation. These aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like. They also maybe formulated as pharmaceuticals for non-pressured preparations, such asin a nebulizer or an atomizer. Such spray formulations also may be usedto spray mucosa.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The compounds and compositions of the invention can be administered in aphysiologically acceptable diluent in a pharmaceutical carrier, such asa sterile liquid or mixture of liquids, including water, saline, aqueousdextrose and related sugar solutions, an alcohol, such as ethanol,isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol orpolyethylene glycol, dimethylsulfoxide, glycerol ketals, such as2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, such aspoly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester orglyceride, or an acetylated fatty acid glyceride with or without theaddition of a pharmaceutically acceptable surfactant, such as a soap ora detergent, suspending agent, such as pectin, carbomers,methylcellulose, hydroxypropylmethylcellulose, orcarboxymethylcellulose, or emulsifying agents and other pharmaceuticaladjuvants.

Oils, which can be used in parenteral formulations include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includeoleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters.

Suitable soaps for use in parenteral formulations include fatty alkalimetal, ammonium, and triethanolamine salts, and suitable detergentsinclude (a) cationic detergents such as, for example, dimethyl dialkylammonium halides, and alkyl pyridinium halides, (b) anionic detergentssuch as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin,ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionicdetergents such as, for example, fatty amine oxides, fatty acidalkanolamides, and polyoxyethylenepolypropylene copolymers, (d)amphoteric detergents such as, for example, alkyl-b-aminopropionates,and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixturesthereof.

Preservatives and buffers may be used. In order to minimize or eliminateirritation at the site of injection, such compositions may contain oneor more nonionic surfactants having a hydrophile-lipophile balance (HLB)of from about 12 to about 17. The quantity of surfactant in suchformulations will typically range from about 5% to about 15% by weight.Suitable surfactants include polyethylene sorbitan fatty acid esters,such as sorbitan monooleate and the high molecular weight adducts ofethylene oxide with a hydrophobic base, formed by the condensation ofpropylene oxide with propylene glycol. The parenteral formulations canbe presented in unit-dose or multi-dose sealed containers, such asampoules and vials, and can be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid excipient,for example, water, for injections, immediately prior to use.Extemporaneous injection solutions and suspensions can be prepared fromsterile powders, granules, and tablets of the kind previously described.

Additionally, the compounds of the invention, or compositions comprisingsuch compounds, can be made into suppositories by mixing with a varietyof bases, such as emulsifying bases or water-soluble bases. Formulationssuitable for vaginal administration can be presented as pessaries,tampons, creams, gels, pastes, foams, or spray formulas containing, inaddition to the active ingredient, such carriers as are known in the artto be appropriate.

The invention also provides a method of diagnosing cancer in anindividual comprising (a) obtaining a sample comprising myeloid cellsfrom the individual; and (b) determining TGFβ receptor II expression inthe myeloid cells, wherein increased TGFβ receptor II expression in themyeloid cells relative to a control indicates a diagnosis of cancer inthe individual.

Expression of TGFβ receptor II mRNA and/or protein can be determined byany suitable method including, but not limited to, PCR (RT-PCR,quantitative RT-PCR), microarrays, Northern blotting, and Westernblotting.

The control can be any suitable control, such as an expression level ofTGFβ receptor II mRNA and/or protein from myeloid cells of a normal(healthy) individual or group of normal (healthy) individuals.

The individual can be any suitable individual, such as a mammalincluding a mouse, rat, hamster, guinea pig, rabbit, cat, dog, pig,horse, cow, or primate (e.g., human).

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

EXAMPLE 1

This example demonstrates the generation of a myeloid-specific TGFβreceptor II knockout mouse.

Myeloid cells play an important role in tumor progression. They suppresshost immune surveillance and influence the tumor microenvironment (see,e.g., Gabrilovich et al., Nat. Rev. Immunol., 9: 162-174 (2009);Pollard, Nat. Rev. Immunol., 9: 259-270 (2009); Mantovani, Nature, 457:36-37 (2009), Fridlender et al., Cancer Cell, 16: 183-194 (2009);Balkwill et al., Nature, 431: 405-406 (2004), Yang et al., Cancer Cell,6: 409-421 (2004); and Yang et al., Cancer Cell, 13: 23-35 (2008)).Myeloid cells are also present in the lungs prior to tumor cell arrivaland contribute to pre-metastatic niche formation and environmentalteration (see Kaplan et al., Nature, 438: 820-827 (2005); and Yan etal., Cancer Res., 70: 6139-6149 (2010)). These include tumor-associatedmacrophages (TAM, Mac-1+ or F4/80+ cells), Gr-1+CD11b+ cells or myeloidderived suppressor cells (MDSCs), and tumor associated neutrophils (TAN,CD11b+Ly6G+ cells). One of the most important properties of these cellsis the increased TGFβ production and increased Th2 polarization (see,e.g., Yang, et al., Cancer Cell, 13: 23-35 (2008); and Flavell et al.,Nat. Rev. Immunol. 10: 554-567 (2010)). However, there are no reports asto how TGFβ signaling in myeloid cells affects tumor phenotype.

To investigate the effect of myeloid specific abrogation of TGFβsignaling on tumor phenotype, knock out mice with a specific deletion ofthe TGFβ receptor II in myeloid cells (Tgfbr2^(MyeKO) were generated.Mice with targeted deletion of Tgfbr2 in myeloid cells) (Tgfbr2^(MyeKO)were generated through the cross breeding of foxed Tgfbr2(Tgfbr2^(flox/flox)) (see, e.g., Chytil et al., Genesis, 32: 73-75(2002); and Forrester et al., Cancer Res., 65: 2296-2302 (2005)) micewith LysM-Cre transgenic mice (in C57BL/6 and 129 background). Miceheterozygous for LysM-Cre and heterozygous for the foxed Tgfbr2 allelewere further bred with wild-type Balb/c or C57BL/6 for 10 generations togenerate a Balb/c or C57BL/6 background. LysM-Cre transgenic mice havebeen well characterized and used in many other studies to delete floxedgenes specifically in myeloid cells, both neutrophils andmonocytes/macrophages (see, e.g., Sinha et al., J. Immunol., 173:1763-1771 (2004); Sun et al., Blood, 104: 3758-3765 (2004); and Hazenboset al., Blood, 104: 2825-2831 (2004)). The Tgfbr2^(MyeKO) mice appearedto be normal with no alteration in hematopoiesis (see FIGS. 1A and 1B).This includes macrophages (CD11b+F4/80+), MDCSs (Gr-1+CD11b+), B cells(B220), CD4+ T cells, CD8+ T cells, as well as NK cells (NK 1.1), andNKT cells (NK1.1 and TCRβ) derived from spleen, thymus, bone marrow andlymph nodes (FIGS. 1A and 1B).

EXAMPLE 2

This example demonstrates that reduced expression of TGFβ signalingreduces lung metastasis.

The 4T1 mammary tumor model shares many characteristics with humanbreast cancer, particularly its ability to spontaneously metastasize tothe lungs. In an orthotopic metastasis design, 5×10⁴ 4T1 cells in 50 μLPBS were injected into the #2 mammary fat pad (MFP) of Tgfbr2^(MyeKO)and floxed control Tgfbr2^(flox/flox) mice. Mice were sacrificed 28 dayslater, and the number and size of lung metastasis was evaluated.Tgfbr2^(MyeKO) mice showed a decreased ability to support tumormetastasis after injection of 4T1 mammary tumor cells (see FIG. 2), withno difference in primary tumor size.

In an experimental metastasis design, 2×10⁵ 4T1 cells were injected intothe tail vein of Tgfbr2^(MyeKO) and foxed control Tgfbr2^(flox/flox)mice. The size of tumors was determined by direct measurement of tumordimensions at 2-3 day intervals using calipers. The number of lungmetastasis was evaluated when the mice were euthanized at day 25 afterinjection. There was a significant reduction in metastasis inTgfbr2^(MyeKO) (see FIG. 3). This result was recapitulated in the LLCexperimental metastasis model in which Tgfbr2^(MyeKO) mice in a C57BL/6background were injected in the tail vein with or 2.5×10⁵ LLC cells andsacrificed at day 21 after injection (see FIG. 4).

These data support that decreased expression of TGFβ signaling resultsin a decrease of lung metastasis in a mouse model.

EXAMPLE 3

This example demonstrates that the inventive methods inhibit metastasis.

To further confirm the inhibitory effect of myeloid-specific Tgfbr2deletion on tumor metastasis, Tgfbr2^(MyeKO) bone marrow (B.M.) wastransplanted into wild-type mice bearing 4T1 tumors. 5×10⁵ 4T1 cellswere injected into the #4 MFP on day 0. To better model clinicalmetastatic disease the primary tumor was surgically removed on day 15and metastasis allowed to continue until day 34 allowing the mice todevelop tumor invasion and metastasis. On day 34, the mice wereirradiated and subjected to B.M. transplantation from eitherTgfbr2Mye^(KO) mice or Tgfbr2^(flox/flox) control mice. Lung metastaseswere examined on day 63 and thereafter (see FIG. 5).

100% survival of the mice that received B.M. from the Tgfbr2^(MyeKO)mice was observed, whereas approximately 55% of the mice that receivedB.M. from Tgfbr2^(f1ox/flox) control mice exhibited decreased survival(see FIG. 6A). In addition, a significantly reduced number of lungmetastases were observed in mice that received Tgfbr2^(MyeKO) B.M.relative to those that received control B.M. (see FIG. 6B).

These data suggest that myeloid-specific TGFβ signaling constitutes anessential part of the metastasis-promoting role of TGFβ. When Tgfbr2 isablated in myeloid cells, tumor metastasis is significantly decreased.Notably, this experimental model uncovers mechanisms different fromthose observed in mice with Tgfbr2 deletion in FSP+ fibroblasts, whichdevelop invasive squamous cell carcinoma in the fore-stomach, andintraepithelial neoplasia in the prostate (see, e.g., Bhowmick et al.,Science, 303: 848-851 (2004)). The consequences of myeloid-specificTgfbr2 ablation are also different from deletion of Smad4, signaling inT cells, which induces gastrointestinal cancer development (see, e.g.,Kim et al., Nature, 441: 1015-1019 (2006)).

Instead, these data are reminiscent of those observed following blockadeof TGFβ signaling in T cells using CD4dnTGF-RII mice, which confersresistance to an EL-4 lymphoma or a B16-F10 melanoma tumor challenge(see, e.g., Gorelik et al., Nat. Med., 7: 1118-1122 (2001)). However,those mice developed an autoimmune pathology that is not seen in thecurrent mouse model. The lack of pathology in the current mouse model islikely due to the fact that myeloid cells are massively expanded undertumor conditions (see FIG. 7). In particular, Gr-1+CD11b+ cells, whichproduce large quantities of TGFβ, constitute the majority of thetumor-associated myeloid cells in the 4T1 mammary tumor and Lewis lungcarcinoma (LLC) models. Splenic Gr-1+CD11b+ cells from tumor-bearingmice expressed significantly higher levels of TGFβ receptor II comparedwith their non-tumor-bearing counterparts.

These data support that the specific deletion of myeloid Tgfbr2 producesa pronounced antitumor effect with very few adverse effects.

EXAMPLE 4

This example demonstrates the correlation between TGFβ receptor IIoverexpression and cancer.

Immature myeloid cells are overproduced in tumor hosts includingpatients with a variety of cancers (see, e.g., Yang et al., Cancer Cell,6: 409-421 (2004); and Almand et al., J. Immunol., 166: 678-689 (2001)).In humans, these cells have been identified as myeloid linage markerCD33+CD34+CD15+ cells (Yang et al., Cancer Cell, 6: 409-421 (2004); Zeaet al., Cancer Res., 65: 3044-3048 (2005); Srivastava et al., CancerImmunol. Immunother., 57: 1493-1504 (2008); Hoechst et al., Hepatology,50: 799-807 (2009); and Chalmin et al., J. Clin. Invest., 120: 457-471(2010)).

To investigate whether TGFβ receptor II was over-expressed in thesehuman myeloid cells, peripheral blood from 16 patients with metastaticnon-small cell lung cancers was collected. CD33+CD34+CD15+ myeloid cellsaccounted for approximately 85% of the total leukocytes in thesepatients, clearly higher than that of healthy individuals (28%, n=4)(see FIG. 8). Sorted immature myeloid cells from cancer patients alsoshowed overtly increased TGFβ receptor II expression compared to thosefrom healthy individuals (see FIGS. 12A-B), suggesting a strong clinicalrelevance for TGFβ receptor II over-expression in myeloid cells.

Furthermore, the expression of Tgfβ1 and TGFβ receptor II mRNA in humanperipheral blood mononuclear cells in cohorts of lung cancer (GSE20189)and breast cancer (GSE27567) was determined. The lung cancer and breastcancer datasets were analyzed by Genespring GX 10.0 software. TGFβreceptor II expression correlated with the degree of malignancy in thelung and breast cancer datasets. These results indicate that TGFβreceptor II expression in monocytes can be used for diagnosis and/orprognosis of cancer.

The over-expression of TGFβ receptor II in both human and mouse myeloidcells from cancer hosts strongly supports that TGFβ receptor IIsignaling in myeloid cells affects tumor progression and metastasis.

EXAMPLE 5

This example demonstrates the characterization of reduced TGFβsignaling.

Gr-1+CD11b+ cells are present in the premetastatic lung (prior to tumorcell arrival). They change the lung into an inflammatory andproliferative environment, diminish immune protection, and promotemetastasis through aberrant vasculature formation. The premetastaticlung is characterized by increased growth factors, inflammatorycytokines, and chemokines, such as the chemokine PF4. PF4, also known asCXCL4, was significantly increased in lungs of 4T1 tumor bearing mice atday 10 and 14 when compared with non-tumor control mice.

PF4 belongs to a CXCL chemokine family that includes CXCL9, CXCL10, andCXCL11. These chemokines signal through CXCR3, a G protein coupledreceptor. Interestingly, other than PF4, there was no change in theexpression of other members of this chemokine family (i.e., CXCL9,CXCL10, and CXCL11). Notably, the deletion of Tgfbr2 decreased PF4expression in myeloid cells (see FIG. 9A) and in the premetastatic lungsof Tgfbr2^(MyeKO) (see FIG. 9B).

To determine the functional significance of decreased PF4/CXR3 signalingin tumor metastasis, CXCR3 knockout mice (see, e.g., Pan et al., J.Immunol., 176: 1456-1464 (2006)) were used. Deletion of CXCR3dramatically decreased the number of lung metastasis in the mice thatreceived 4T1 tumor injection in the #2 MFP (see FIG. 9C) with no effecton primary tumor size or weight (see FIG. 9D).

These data support that PF4/CXCR3 chemokine axis plays a specific andcritical role in 4T1 tumor lung metastasis.

EXAMPLE 6

This example demonstrates the further characterization of reduced TGFβsignaling.

TGFβ signaling is a critical mediator of polarization of myeloid cells.Therefore, Th1/Th2 cytokine expression of Gr-1+CD11b+ cells wasexamined. Interestingly, the expression of Th2 type cytokines, includingIL-10 and IL4, was reduced in myeloid cells with the Tgfbr2 deletioncompared to controls, with no difference in Th1 type cytokine production(e.g., IL-12 and IFNγ) (see FIG. 10A). There also was a reduction inarginase and iNOS levels (see FIG. 10A), which are implicated in theimmune suppression effects of Gr-1+CD11b+ cells. These results werefurther confirmed with a cytokine protein array assay (see FIG. 10B).

Since tumor associated myeloid cells exert immune suppression throughinhibiting multiple immune cell function in tumor hosts, whethermyeloid-specific Tgfbr2 deletion resulted in an improved function ofCD4, CD8, B, NK, or macrophage cells was examined. Single cellsuspensions from spleen were made, and IFNγ ELISPOT and intracellularcytokine staining of IL2, IL-10, IL4 (CD4 T cells), IFNγ, IL2 (CD8 Tcells), CD69, 41BB (B cell function), IFNγ (NK cell function), as wellas IL12 and IL-10 (macrophage function) were performed betweenTgfbr2^(MyeKO) and the control littermates.

An increased percentage of IFN-γ positive CD8+ T cells was observed inthe spleen of tumor-bearing Tgfbr2^(MyeKO) mice compared toTgfbr2^(flox/flox) mice (see FIG. 11A). No difference was found in othercytokines or cell types. This was consistent with the increased numberof IFN-γ producing cells detected by ELISPOT in the spleens ofTgfbr2^(MyeKO) mice (see FIG. 11B). Importantly, systemic neutralizationof IFN-γ diminished the inhibitory effect of myeloid Tgfbr2 deletion onmetastasis (see FIG. 11C) with no effect on tumor size (see FIG. 11D).

This data suggests that genetic inactivation of TGFβ receptor II inmyeloid cells likely decreases immune suppression in tumor bearing hoststhrough improved Th1/Th2 balance and elevated IFNγ production in CD8 Tcells, which likely contributes to reduced tumor metastasis inTgfbr2^(MyeKO) mice.

Gr-1+CD11b+ cells are one of the major sources of TGFβ in the tumorbearing host. Deletion of Tgfbr2 decreased TGFβ1 production inGr-1+CD11b+ cells (see FIG. 11E), suggesting possible autocrine and/orparacrine loops that enhance TGFβ production and signaling in myeloidcells. It is not clear whether TGFβ production directly converts myeloidcells from an M1 to M2 phenotype or is the result of M2 TAMpolarization. The data show that deletion of myeloid Tgfbr2 induced adecrease of both Th2 cytokines and TGFβ1 production, which wasassociated with increased IFN-γ expression in CD8 T cells. This likelyimproves the host immune surveillance in the Tgfbr2^(MyeKO) mice. Takentogether, these studies demonstrate that myeloid-specific TGFβ signalingis a significant part of the tumor-promoting effects of TGFβ, andprovides a therapeutic opportunity for new approaches to cancer therapy.

EXAMPLE 7

This example demonstrates the mechanism of reduced TGFβ signaling.

To determine the mechanisms underlying decreased Th2 cytokine or PF4expression in myeloid cells of Tgfbr2^(MyeKO) mice, the expression ofpotentially relevant genes was determined. Increased expression ofglycogen synthase kinase 3 (GSK3) and NFκB was observed in Gr-1+CD11b+cells sorted from Tgfbr2^(MyeKO) mice. GSK3 is a serine/threonineprotein kinase that mediates the addition of phosphate molecules intoserine and threonine amino acid residues. GKS3 has been implicated inthe production of inflammation-associated cytokines (see, e.g., Park etal., Nat. Immunol., 12: 607-615 (2011); and Woodgett et al., Nat.Immunol., 6: 751-752 (2005)).

Gr-1+CD11b+ myeloid cells were sorted with MACS from Tgfbr2^(MyeKO) andcultured for 6 hours with an inhibitor of GSK3 (SB216763, Sigma) or PI3K(LY294002, Calbiochem) at different doses (2, 5, and 10 μM for the GSK3inhibitor; 5, 10, and 20 μM for the PI3K inhibitor). SB216763 is apotent and selective ATP-competitive inhibitor of the serine/threonineprotein kinase GSK α and β. LY294002 competitively inhibits ATP bindingof the catalytic subunit of P13 kinases, consequently enhancing GSK3activity. GSK3 Western blotting was performed to detect inhibitingactivity. The expression of IL-10, IL-4, and PF4 was examined usingWestern blotting and quantitative PCR.

The GSK3-specific inhibitor (SB216763) reversed the down-regulation ofIL-10, IL-4, and PF4 in the myeloid cells lacking Tgfbr2 at both themRNA level and protein level. The inhibitor of PI3K (LY294002), theupstream mediator of GSK3, resulted in the inverse effect of GSK3inhibition (i.e., decreased IL-10, IL4, and P4). This is consistent witha negative regulatory role of PI3K on GSK3 that promotes inhibitoryphosphorylation of GSK3. Inhibition of NFκB with a specific inhibitor(BMS-345541) did not show a significant effect. These data indicate thatTGFβ regulation of Th2 and PF4 likely is mediated by the PI3K and GSK3signaling pathways. Therefore, P13K inhibitors (e.g., LY294002) and GSK3enhancers can be used to target or modify myeloid cells in cancerpatients to inhibit cancer and/or metastasis.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A method of inhibiting metastasis in a cancer patient comprisingreducing TGFβ receptor II expression in myeloid cells in the cancerpatient.
 2. The method of claim 1, wherein the TGFβ receptor IIexpression is reduced using RNA interference (RNAi).
 3. The method ofclaim 1, wherein the TGFβ receptor II expression is reduced by mutatingthe nucleic acid sequence encoding TGFβ receptor II in myeloid cells. 4.The method of claim 1, wherein the TGFβ receptor II expression isreduced by knocking out the nucleic acid sequence encoding TGFβ receptorII in myeloid cells.
 5. The method of claim 1, wherein the myeloid cellsare removed from the cancer patient prior to the reduction in TGFβreceptor II expression.
 6. The method of claim 5, wherein the myeloidcells are administered to the cancer patient following the reduction inTGFβ receptor II expression.
 7. A method of inhibiting metastasis in acancer patient comprising: (a) removing bone marrow comprising myeloidcells from the cancer patient, (b) reducing TGFβ signaling in themyeloid cells of the bone marrow ex vivo to yield TGFβsignaling-deficient bone marrow, and (c) administering the TGFβsignaling-deficient bone marrow to the cancer patient, so as to inhibitmetastasis in the cancer patient.
 8. The method of claim 7, wherein TGFβsignaling is reduced by reducing TGFβ receptor II expression in myeloidcells.
 9. The method of claim 7, wherein the TGFβ signaling is reducedusing RNA interference (RNAi).
 10. The method of claim 9, wherein theTGFβ signaling is reduced by mutating the nucleic acid sequence encodingTGFβ receptor II in myeloid cells.
 11. The method of claim 9, whereinthe TGFβ signaling is reduced by knocking out the nucleic acid sequenceencoding TGFβ receptor II in myeloid cells.
 12. A method of inhibitingmetastasis in a cancer patient comprising transplanting TGFβ receptorII-deficient myeloid cells to the cancer patient.
 13. The method ofclaim 1, wherein the cancer patient is an animal.
 14. The method ofclaim 13, wherein the cancer patient is a human.
 15. A vector comprisinga TGFβ receptor II RNAi nucleic acid sequence operably linked to amyeloid-specific promoter.
 16. The vector of claim 15, wherein thevector is a viral vector, a plasmid, a yeast, or a nanoparticle.
 17. Amethod of diagnosing cancer in an individual comprising (a) obtaining asample comprising myeloid cells from the individual; and (b) determiningTGFβ receptor II expression in the myeloid cells, wherein increased TGFβreceptor II expression in the myeloid cells relative to a controlindicates a diagnosis of cancer in the individual.
 18. A method ofmodulating TGFβ activity in myeloid cells in a cancer patient comprisingadministering a regulator of at least one of the GSK3 and PI3K pathwaysto the patient.
 19. The method of claim 18, wherein TGFβ activity inmyeloid cells is reduced by administering an inhibitor of PI3K or anenhancer of GSK3.
 20. The method of claim 18, wherein the PI3K inhibitoris LY294002.